Targeting the hydrophilic regions of recombinant proteins by MS via in-solution buffer-free trypsin digestion

Abstract

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with “high-mannose” N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

The SARS-CoV-2 receptor-binding domain expressed in Pichia pastoris as a candidate vaccine antigen

“…The remaining deglycosylated protein was digested by using an in-solution buffer-free trypsin digestion protocol previously reported [20] and adapted to the analysis of SARS-…”

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

“…Recently, the principles of the in-gel buffer-free digestion protocol [19] were extended to in-solution buffer-free digestion (BFD) of other proteins [20]. In-solution BFD protocol improved the sequence coverage of certain regions of proteins represented by short and hydrophilic peptides including some N-glycopeptides, short peptides linked by disulfide bonds, and hydrophilic His 6 tag C-terminal peptides [20].…”

“…In this work, we adapted the in-solution BFD protocol [20] to the analysis of the products of six SARS-CoV-2 RBD expression constructs from five different expression systems. The implemented in-solution BFD method avoids buffers and desalting is carried out by protein precipitation, allowing very high sequence coverage (≥ 99%) and the detection of PTMs including those located at the N-and the C-terminal end.…”

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

“…Recently, the principles of the in-gel buffer-free digestion protocol (23) were extended to insolution buffer-free digestion (BFD) of other proteins (24). In-solution BFD protocol improved the sequence coverage of certain regions of proteins represented by short and hydrophilic peptides including some N-glycopeptides, short peptides linked by disulfide bonds as well as hydrophilic C-terminal peptides of proteins that contain a tandem repeat of six histidine residues (24).…”

“…Considering that subunit vaccines based on the recombinant RBD are very well-represented among the different strategies for the development of vaccines against COVID-19, in this work, we adapted the in-solution BFD protocol (24) to the analysis of the products of six gene constructs containing the RBD sequence from SARS-CoV-2 obtained in five different expression systems, such as mammalian cells (CHO and HEK-293T), yeast (P. pastoris), bacteria (E. coli) and fungus (Thermothelomyces heterothallica). Unlike the standard protocol that uses salt buffers and desalting through reverse phase microcolumns, the implemented BFD method avoids buffers and desalting is carried out by precipitation, allowing very high sequencecoverage (≥ 99 %) of these RBDs, and the detection of PTMs including those located at the Nand the C-terminal end.…”